Novel human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/282,036, which was filed on Apr. 6, 2001, and isherein incorporated by reference in its entirety.

INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosedpolynucleotides, antagonists and agonists of the proteins, and othercompounds that modulate the expression or activity of the proteinsencoded by the disclosed polynucleotides, which can be used fordiagnosis, drug screening, clinical trial monitoring, the treatment ofdiseases and disorders, and cosmetic or nutriceutical applications.

BACKGROUND OF THE INVENTION

[0003] Kinases mediate the phosphorylation of a wide variety of proteinsand compounds in the cell. Along with phosphatases, kinases are involvedin a range of regulatory pathways. Given the physiological importance ofkinases, they have been subject to intense scrutiny and are proven drugtargets.

SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins andthe corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPs), described for the first time herein, sharestructural similarity with animal kinases, including, but not limitedto, serine-threonine kinases, and carbon catabolite depressing kinases.Accordingly, the described NHPs encode novel kinases having homologuesand orthologs across a range of phyla and species.

[0005] The novel human polynucleotides described herein encode openreading frames (ORFs) encoding proteins of 778, 762, and 703 amino acidsin length (see respectively SEQ ID NOS:2, 4, and 6).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHPs, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cell”) lines that contain gene trap mutations in a murine homologof at least one of the described NHPs. When the unique NHP sequencesdescribed in SEQ ID NOS:1-6 are “knocked-out” they provide a method ofidentifying phenotypic expression of the particular gene, as well as amethod of assigning function to previously unknown genes. In addition,animals in which the unique NHP sequences described in SEQ ID NOS:1-6are “knocked-out” provide a unique source in which to elicit antibodiesto homologous and orthologous proteins, which would have been previouslyviewed by the immune system as “self” and therefore would have failed toelicit significant antibody responses. To these ends, gene trappedknockout ES cells have been generated in murine homologs of thedescribed NHPs.

[0007] Additionally, the unique NHP sequences described in SEQ IDNOS:1-6 are useful for the identification of protein coding sequences,and mapping a unique gene to a particular chromosome. These sequencesidentify biologically verified exon splice junctions, as opposed tosplice junctions that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists of, NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP products, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of the novel humanORFs encoding the described novel human kinase proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHPs described for the first time herein are novel proteinsthat are expressed in, inter alia, human cell lines and human brain,pituitary, cerebellum, spinal cord, lymph node, testis, adrenal gland,uterus, mammary gland, rectum, hypothalamus, ovary, fetal kidney, fetallung, aorta, 6-, 9-, and 12-week old embryos, adenocarcinoma,osteosarcoma, embryonic carcinoma, and normal umbilical vein cells. Thedescribed sequences were compiled from sequences available in GENBANK,and cDNAs generated from human adult and fetal brain, pituitary,hypothalamus, testis, and fetus mRNAs (Edge Biosystems, Gaithersburg,Md.) that were identified using primers generated from human genomicDNA.

[0011] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described polynucleotides, includingthe specifically described NHPs, and the NHP products; (b) nucleotidesthat encode one or more portions of a NHP that correspond to anyfunctional domain(s), and the polypeptide products specified by suchnucleotide sequences, including, but not limited to, the novel regionsof any active domain(s); (c) isolated nucleotides that encode mutantversions, engineered or naturally occurring, of the described NHPs, inwhich all or a part of at least one domain is deleted or altered, andthe polypeptide products specified by such nucleotide sequences,including, but not limited to, soluble proteins and peptides; (d)nucleotides that encode chimeric fusion proteins containing all or aportion of a coding region of a NHP, or one of its domains (e.g., areceptor/ligand binding domain, accessory protein/self-associationdomain, etc.) fused to another peptide or polypeptide; or (e)therapeutic or diagnostic derivatives of the described polynucleotides,such as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA,or gene therapy constructs, comprising a sequence first disclosed in theSequence Listing.

[0012] As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1x SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2x SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), yet still encode a functionally equivalent NHPproduct. Functional equivalents of a NHP include naturally occurringNHPs present in other species, and mutant NHPs, whether naturallyoccurring or engineered (by site directed mutagenesis, gene shuffling,directed evolution as described in, for example, U.S. Pat. Nos.5,837,458 or 5,723,323, both of which are herein incorporated byreference). The invention also includes degenerate nucleic acid variantsof the disclosed NHP polynucleotide sequences.

[0013] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NOS:1, 3 or 5 (as measured by BLAST sequencecomparison analysis using, for example, the GCG sequence analysispackage, as described herein, using default parameters).

[0014] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP-encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed herein. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80 bases long, or about34 to about 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0015] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a microarray or high-throughput“chip” format). Additionally, a series of NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS:1-6 can be used as a hybridization probe, inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS:1-6, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon, are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405,the disclosures of which are herein incorporated by reference in theirentirety.

[0016] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-6 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is usually within a range of between about 8to about 2000 nucleotides. Preferably the probes consist of 60nucleotides, and more preferably 25 nucleotides, from the sequencesfirst disclosed in SEQ ID NOS:1-6.

[0017] For example, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length, can partially overlap each other, and/orthe sequence may be represented using oligonucleotides that do notoverlap. Accordingly, the described polynucleotide sequences shalltypically comprise at least about two or three distinct oligonucleotidesequences of at least about 8 nucleotides in length that are each firstdisclosed in the described Sequence Listing. Such oligonucleotidesequences can begin at any nucleotide present within a sequence in theSequence Listing, and proceed in either a sense (5′-to-3′) orientationvis-a-vis the described sequence or in an antisense orientation.

[0018] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions, andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-6 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel components,or gene functions that manifest themselves as novel phenotypes.

[0019] Probes consisting of sequences first disclosed in SEQ ID NOS:1-6can also be used in the identification, selection, and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets, andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the intended target of thedrug. These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0020] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-6 can be utilized in microarrays, or other assay formats, toscreen collections of genetic material from patients who have aparticular medical condition. These investigations can also be carriedout using the sequences first disclosed in SEQ ID NOS:1-6 in silico, andby comparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0021] Thus the sequences first disclosed in SEQ ID NOS:1-6 can be usedto identify mutations associated with a particular disease, and also indiagnostic or prognostic assays.

[0022] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence, inconjunction with the presence of one or more specific oligonucleotidesequence(s) first disclosed in SEQ ID NOS:1-6. Alternatively, arestriction map specifying the relative positions of restrictionendonuclease digestion sites, or various palindromic or other specificoligonucleotide sequences, can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich., etc.), can optionally be used in conjunction with one or morediscrete nucleotide sequence(s) present in the sequence that can bedescribed by the relative position of the sequence relative to one ormore additional sequence(s) or one or more restriction sites present inthe disclosed sequence.

[0023] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6x SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP antisense molecules, useful, for example, inNHP gene regulation and/or as antisense primers in amplificationreactions of NHP nucleic acid sequences. With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0024] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5,-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0025] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0026] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0027] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0028] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

[0029] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (and periodic updates thereof), and Ausubel et al.,1989, supra.

[0030] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0031] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0032] Further, a NHP homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, genomic DNA and/or cDNA obtained byreverse transcription of mRNA prepared from, for example, human ornon-human cell lines or tissue known to express, or suspected ofexpressing, an allele of a NHP gene.

[0033] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0034] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown to express, or suspected of expressing, a NHP gene). A reversetranscription (RT) reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see, e.g., Sambrooket al., 1989, supra.

[0035] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known to express, or suspected of expressing, a NHP, in anindividual putatively carrying a mutant NHP allele, and by extending thenew strand with reverse transcriptase. The second strand of the cDNA isthen synthesized using an oligonucleotide that hybridizes specificallyto the 5′ end of the normal sequence. Using these two primers, theproduct is then amplified via PCR, optionally cloned into a suitablevector, and subjected to DNA sequence analysis through methodswell-known to those of skill in the art. By comparing the DNA sequenceof the mutant NHP allele to that of a corresponding normal NHP allele,the mutation(s) responsible for the loss or alteration of function ofthe mutant NHP gene product can be ascertained.

[0036] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of carrying, or known to carry, amutant NHP allele (e.g., a person manifesting a NHP-associated phenotypesuch as, for example, behavioral disorders, immune disorders, obesity,high blood pressure, etc.), or a cDNA library can be constructed usingRNA from a tissue known to express, or suspected of expressing, a mutantNHP allele. A normal NHP gene, or any suitable fragment thereof, canthen be labeled and used as a probe to identify the corresponding mutantNHP allele in such libraries. Clones containing mutant NHP sequences canthen be purified and subjected to sequence analysis according to methodswell-known to those skilled in the art.

[0037] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.).

[0038] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to a NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0039] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721, 5,837,458, 6,117,679, and 5,723,323, which areherein incorporated by reference in their entirety.

[0040] The invention also encompasses: (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators, and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40 oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0041] Where, as in the present instance, some of the described NHPpeptides or polypeptides are thought to be cytoplasmic or nuclearproteins (although processed forms or fragments can be secreted ormembrane associated), expression systems can be engineered that producesoluble derivatives of a NHP (corresponding to a NHP extracellularand/or intracellular domains, or truncated polypeptides lacking one ormore hydrophobic domains) and/or NHP fusion protein products (especiallyNHP-Ig fusion proteins, i.e., fusions of a NHP domain to an IgFc), NHPantibodies, and anti-idiotypic antibodies (including Fab fragments) thatcan be used in therapeutic applications. Preferably, the aboveexpression systems are engineered to allow the desired peptide orpolypeptide to be recovered from the culture media.

[0042] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0043] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs, or inappropriately expressed NHPs, for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

[0044] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto NHPs, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of a soluble NHP, a NHP-IgFc fusion protein, or ananti-idiotypic antibody (or its Fab) that mimics the NHP, could activateor effectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules, can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

[0045] Various aspects of the invention are described in greater detailin the subsections below.

THE NHP SEQUENCES

[0046] The cDNA sequences (SEQ ID NOS:1, 3, and 5) and correspondingdeduced amino acid sequences (SEQ ID NOS:2, 4, and 6, respectively) ofthe described NHPs are presented in the Sequence Listing.

[0047] Expression analysis has provided evidence that the described NHPscan be expressed in a moderate range of human tissues. In addition toserine-threonine kinases, the described NHPs also share significantsimilarity to a range of additional kinase families, including kinasesassociated with signal transduction, from a variety of phyla andspecies. The genomic locus encoding the described NHPs is apparentlyencoded on human chromosome 19 (see GENBANK accession no. AC020922). Assuch, the described sequences are useful, inter alia, for mapping thecoding regions of the human genome, and particularly chromosome 19.

[0048] Given the physiological importance of protein kinases, they havebeen subject to intense scrutiny, as exemplified and discussed in U.S.Pat. Nos. 5,756,289 and 5,817,479, herein incorporated by reference intheir entirety, which describe uses and utilities that are applicable tothe described NHPs.

[0049] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees, may be usedto generate NHP transgenic animals.

[0050] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

[0051] The present invention provides for transgenic animals that carrya NHP transgene in all their cells, as well as animals that carry atransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. A transgene may be integrated as asingle transgene, or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. A transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

[0052] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0053] The transgene can also be selectively introduced into aparticular cell-type, thus inactivating the endogenous NHP gene in onlythat cell-type, by following, for example, the teaching of Gu et al.,1994, Science 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

[0054] Once transgenic animals have been generated, the expression ofthe recombinant NHP sequence may be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals mayalso be assessed using techniques that include, but are not limited to,Northern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of NHP gene-expressingtissue may also be evaluated immunocytochemically using antibodiesspecific for the NHP transgene product.

[0055] The present invention also provides for “knock-in” animals.Knock-in animals are those in which a polynucleotide sequence (i.e., agene or a cDNA) that the animal does not naturally have in its genome isinserted in such a way that it is expressed. Examples include, but arenot limited to, a human gene or cDNA used to replace its murine orthologin the mouse, a murine cDNA used to replace the murine gene in themouse, and a human gene or cDNA or murine cDNA that is tagged with areporter construct used to replace the murine ortholog or gene in themouse. Such replacements can occur at the locus of the murine orthologor gene, or at another specific site. Such knock-in animals are usefulfor the in vivo study, testing and validation of, intra alia, human drugtargets, as well as for compounds that are directed at the same, andtherapeutic proteins.

NHPS AND NHP POLYPEPTIDES

[0056] NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto a NHP, and as reagents in assays for screening for compounds that canbe used as pharmaceutical reagents useful in the therapeutic treatmentof mental, biological, or medical disorders and diseases. Given thesimilarity information and expression data, the described NHPs can betargeted (by drugs, oligonucleotides, antibodies, etc.) in order totreat disease, or to therapeutically augment the efficacy of therapeuticagents.

[0057] The Sequence Listing discloses the amino acid sequences encodedby the described NHP-encoding polynucleotides. The NHPs displayinitiator methionines that are present in DNA sequence contextsconsistent with eucaryotic translation initiation sites. The NHPs do notdisplay consensus signal sequences, which indicates that they may becytoplasmic or possibly nuclear proteins, although they may also besecreted or membrane associated (given the presence of severalhydrophobic domains).

[0058] The NHP amino acid sequences of the invention include the aminoacid sequences presented in the Sequence Listing, as well as analoguesand derivatives thereof. Further, corresponding NHP homologues fromother species are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described herein are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference), are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0059] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences, as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHP substrate, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,phosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described herein, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0060] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where the NHPpeptide or polypeptide can exist, or has been engineered to exist, as asoluble or secreted molecule, the soluble NHP peptide or polypeptide canbe recovered from the culture media. Such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ. Purification or enrichment of a NHP from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well-known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of a NHP, but to assess biological activity, e.g., incertain drug screening assays.

[0061] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0062] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing a NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in-frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. PGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

[0063] In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreignpolynucleotide sequences. The virus grows in Spodoptera frugiperdacells. A NHP coding sequence can be cloned individually into anon-essential region (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of a NHP coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted sequence isexpressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S.Pat. No. 4,215,051).

[0064] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, may be provided. Furthermore, theinitiation codon should be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al., 1987, Methods in Enzymol. 153:516-544).

[0065] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for the desired processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0066] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHP sequences described herein can be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then switched to a selective media. The selectable marker inthe recombinant plasmid confers resistance to the selection and allowscells to stably integrate the plasmid into their chromosomes and grow toform foci, which in turn can be cloned and expanded into cell lines.This method may advantageously be used to engineer cell lines thatexpress the NHP product. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of the NHP product.

[0067] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine, phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0068] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.Another exemplary system allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the sequence of interest is subcloned into a vaccinia recombinationplasmid such that the sequence's open reading frame is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²⁺·nitriloacetic acid-agarose columns, and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

[0069] Also encompassed by the present invention are fusion proteinsthat direct a NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching an appropriate signal sequence to a NHPwould also transport a NHP to a desired location within the cell.Alternatively targeting of a NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,R.R.C., ed., Oxford University Press, N.Y., and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures, which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of NHPs to a target site ordesired organ, where they cross the cell membrane and/or the nucleuswhere the NHPs can exert their functional activity. This goal may beachieved by coupling of a NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. Provisional Patent Application Ser. Nos. 60/111,701and 60/056,713, both of which are herein incorporated by reference, forexamples of such transducing sequences), to facilitate passage acrosscellular membranes, and can optionally be engineered to include nuclearlocalization signals.

[0070] Additionally contemplated are oligopeptides that are modeled onan amino acid sequence first described in the Sequence Listing. Such NHPoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHP oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

[0071] The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components which naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide, or by chemically synthesizing theprotein or polypeptide.

[0072] Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

ANTIBODIES TO NHP PRODUCTS

[0073] Antibodies that specifically recognize one or more epitopes of aNHP, epitopes of conserved variants of a NHP, or peptide fragments of aNHP, are also encompassed by the invention. Such antibodies include, butare not limited to, polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0074] The antibodies of the invention can be used, for example, in thedetection of a NHP in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of a NHP. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes for the evaluation of the effect of test compounds onexpression and/or activity of a NHP expression product. Additionally,such antibodies can be used in conjunction with gene therapy to, forexample, evaluate normal and/or engineered NHP-expressing cells prior totheir introduction into a patient. Such antibodies may additionally beused in methods for the inhibition of abnormal NHP activity. Thus, suchantibodies may be utilized as a part of treatment methods.

[0075] For the production of antibodies, various host animals may beimmunized by injection with a NHP, a NHP peptide (e.g., onecorresponding to a functional domain of a NHP), truncated NHPpolypeptides (NHP in which one or more domains have been deleted),functional equivalents of a NHP, or mutated variants of a NHP. Such hostanimals may include, but are not limited to, pigs, rabbits, mice, goats,and rats, to name but a few. Various adjuvants may be used to increasethe immunological response, depending on the host species, including,but not limited to, Freund's adjuvant (complete and incomplete), mineralsalts such as aluminum hydroxide or aluminum phosphate, chitosan,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Alternatively, the immune response could be enhanced bycombination and/or coupling with molecules such as keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin,or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0076] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole. et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof.The hybridomas producing the mAbs of this invention may be cultivated invitro or in vivo. Production of high titers of mAbs in vivo makes thisthe presently preferred method of production.

[0077] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger. et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454), by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity, canbe used. A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine mAb and a human immunoglobulinconstant region. Such technologies are described in U.S. Pat. Nos.6,114,598, 6,075,181 and 5,877,397 and their respective disclosures,which are herein incorporated by reference in their entirety. Alsoencompassed by the present invention is the use of fully humanizedmonoclonal antibodies, as described in U.S. Pat. No. 6,150,584 andrespective disclosures, which are herein incorporated by reference intheir entirety.

[0078] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0079] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of an antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0080] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of a NHP to its cognatereceptor/ligand can be used to generate anti-idiotypes that “mimic” theNHP and, therefore, bind, activate, or neutralize a NHP, NHP receptor,or NHP ligand. Such anti-idiotypic antibodies or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens involving aNHP-mediated pathway.

[0081] Additionally given the high degree of relatedness of mammalianNHPS, the presently described knock-out mice (having never seen a NHP,and thus never been tolerized to a NHP) have a unique utility, as theycan be advantageously applied to the generation of antibodies againstthe disclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

[0082] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 6 1 2337 DNA homo sapiens 1 atgtcgtccg gggccaagga gggaggtgggggctctcccg cctaccacct cccccacccc 60 cacccccacc caccccagca cgcccaatatgtgggcccct atcggctgga gaagacgctg 120 ggcaaaggac agacagggct ggttaaactcggggtccact gcatcacggg tcagaaggtc 180 gccatcaaga tcgtgaaccg ggagaagctgtcggagtcgg tgctgatgaa ggtggagcgg 240 gagatcgcca tcctgaagct catcgaacacccacatgtcc tcaagctcca cgacgtctac 300 gagaacaaga aatatttgta cctggttctggagcacgtct cggggggtga gctattcgac 360 tacctggtaa agaaggggag actgacgcccaaggaggccc gaaagttctt ccgccagatt 420 gtgtctgcgc tggacttctg ccacagctactccatctgcc acagagacct aaagcccgag 480 aacctgcttt tggatgagaa aaacaacatccgcattgcag acttcggcat ggcgtccctg 540 caggtggggg acagcctcct ggagaccagctgcgggtccc cccattatgc gtgtccagag 600 gtgattaagg gggaaaaata tgatggccgccgggcagaca tgtggagctg tggagtcatc 660 ctcttcgccc tgctcgtggg ggctctgccctttgatgacg acaacctccg ccagctgctg 720 gagaaggtga aacggggcgt cttccacatgccccacttca ttcctccaga ttgccagagc 780 ctcctgaggg gaatgatcga agtggagcccgaaaaaaggc tcagtctgga gcaaattcag 840 aaacatcctt ggtacctagg cgggaaacacgagccagacc cgtgcctgga gccagcccct 900 ggccgccggg tagccatgcg gagcctgccatccaacggag agctggaccc cgacgtccta 960 gagagcatgg catcactggg ctgcttcagggaccgcgaga ggctgcatcg cgagctgcgc 1020 agtgaggagg agaaccaaga aaagatgatatattatctgc ttttggatcg gaaggagcgg 1080 tatcccagct gtgaggacca ggacctgcctccccggaatg atgttgaccc cccccggaag 1140 cgtgtggatt ctcccatgct gagccgtcacgggaagcggc gaccagagcg gaagtccatg 1200 gaagtcctga gcatcaccga tgccgggggtggtggctccc ctgtacccac ccgacgggcc 1260 ttggagatgg cccagcacag ccagagatcccgtagcgtca gtggagcctc cacgggtctg 1320 tcctccagcc ctctaagcag cccaaggagtccggtctttt ccttttcacc ggagccgggg 1380 gctggagatg aggctcgagg cgggggctccccgacttcca aaacgcagac gctgccttct 1440 cggggcccca ggggtggggg cgccggggagcagcccccgc cccccagtgc ccgctccaca 1500 cccctgcccg gccccccagg ctccccgcgctcctctggcg ggaccccctt gcactcgcct 1560 ctgcacacgc cccgggccag tcccaccgggaccccgggga caacaccacc ccccagcccc 1620 ggcggtggcg tcgggggagc cgcctggaggagtcgtctca actccatccg caacagcttc 1680 ctgggctccc ctcgctttca ccggcgcaagatgcaggtcc ctaccgctga ggagatgtcc 1740 agcttgacgc cagagtcctc cccggagctggcaaaacgct cctggttcgg gaacttcatc 1800 tccttggaca aagaagaaca aatattcctcgtgctaaagg acaaacctct cagcagcatc 1860 aaagcagaca tcgtccatgc ctttctgtcgatccccagcc tgagtcacag tgtgctgtca 1920 cagaccagct tcagggccga gtacaaggccagtggcggcc cctccgtctt ccaaaagccc 1980 gtccgcttcc aggtggacat cagctcctctgagggtccag agccctcccc gcgacgggac 2040 ggcagcggag gtggtggcat ctactccgtcaccttcactc tcatctcggg tcccagccgt 2100 cggttcaagc gagtggtgga gaccatccaggcacagctcc tgagcactca tgaccagccc 2160 tccgtgcagg ccctggcaga cgagaagaacggggcccaga cccggcctgc tggtgcccca 2220 ccccgaagcc tgcagccccc acccggccgcccagacccag agctgagcag ctctccccgc 2280 cgaggccccc ccaaggacaa gaagctcctggccaccaacg ggacccctct gccctga 2337 2 778 PRT homo sapiens 2 Met Ser SerGly Ala Lys Glu Gly Gly Gly Gly Ser Pro Ala Tyr His 1 5 10 15 Leu ProHis Pro His Pro His Pro Pro Gln His Ala Gln Tyr Val Gly 20 25 30 Pro TyrArg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly Leu Val 35 40 45 Lys LeuGly Val His Cys Ile Thr Gly Gln Lys Val Ala Ile Lys Ile 50 55 60 Val AsnArg Glu Lys Leu Ser Glu Ser Val Leu Met Lys Val Glu Arg 65 70 75 80 GluIle Ala Ile Leu Lys Leu Ile Glu His Pro His Val Leu Lys Leu 85 90 95 HisAsp Val Tyr Glu Asn Lys Lys Tyr Leu Tyr Leu Val Leu Glu His 100 105 110Val Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly Arg Leu 115 120125 Thr Pro Lys Glu Ala Arg Lys Phe Phe Arg Gln Ile Val Ser Ala Leu 130135 140 Asp Phe Cys His Ser Tyr Ser Ile Cys His Arg Asp Leu Lys Pro Glu145 150 155 160 Asn Leu Leu Leu Asp Glu Lys Asn Asn Ile Arg Ile Ala AspPhe Gly 165 170 175 Met Ala Ser Leu Gln Val Gly Asp Ser Leu Leu Glu ThrSer Cys Gly 180 185 190 Ser Pro His Tyr Ala Cys Pro Glu Val Ile Lys GlyGlu Lys Tyr Asp 195 200 205 Gly Arg Arg Ala Asp Met Trp Ser Cys Gly ValIle Leu Phe Ala Leu 210 215 220 Leu Val Gly Ala Leu Pro Phe Asp Asp AspAsn Leu Arg Gln Leu Leu 225 230 235 240 Glu Lys Val Lys Arg Gly Val PheHis Met Pro His Phe Ile Pro Pro 245 250 255 Asp Cys Gln Ser Leu Leu ArgGly Met Ile Glu Val Glu Pro Glu Lys 260 265 270 Arg Leu Ser Leu Glu GlnIle Gln Lys His Pro Trp Tyr Leu Gly Gly 275 280 285 Lys His Glu Pro AspPro Cys Leu Glu Pro Ala Pro Gly Arg Arg Val 290 295 300 Ala Met Arg SerLeu Pro Ser Asn Gly Glu Leu Asp Pro Asp Val Leu 305 310 315 320 Glu SerMet Ala Ser Leu Gly Cys Phe Arg Asp Arg Glu Arg Leu His 325 330 335 ArgGlu Leu Arg Ser Glu Glu Glu Asn Gln Glu Lys Met Ile Tyr Tyr 340 345 350Leu Leu Leu Asp Arg Lys Glu Arg Tyr Pro Ser Cys Glu Asp Gln Asp 355 360365 Leu Pro Pro Arg Asn Asp Val Asp Pro Pro Arg Lys Arg Val Asp Ser 370375 380 Pro Met Leu Ser Arg His Gly Lys Arg Arg Pro Glu Arg Lys Ser Met385 390 395 400 Glu Val Leu Ser Ile Thr Asp Ala Gly Gly Gly Gly Ser ProVal Pro 405 410 415 Thr Arg Arg Ala Leu Glu Met Ala Gln His Ser Gln ArgSer Arg Ser 420 425 430 Val Ser Gly Ala Ser Thr Gly Leu Ser Ser Ser ProLeu Ser Ser Pro 435 440 445 Arg Ser Pro Val Phe Ser Phe Ser Pro Glu ProGly Ala Gly Asp Glu 450 455 460 Ala Arg Gly Gly Gly Ser Pro Thr Ser LysThr Gln Thr Leu Pro Ser 465 470 475 480 Arg Gly Pro Arg Gly Gly Gly AlaGly Glu Gln Pro Pro Pro Pro Ser 485 490 495 Ala Arg Ser Thr Pro Leu ProGly Pro Pro Gly Ser Pro Arg Ser Ser 500 505 510 Gly Gly Thr Pro Leu HisSer Pro Leu His Thr Pro Arg Ala Ser Pro 515 520 525 Thr Gly Thr Pro GlyThr Thr Pro Pro Pro Ser Pro Gly Gly Gly Val 530 535 540 Gly Gly Ala AlaTrp Arg Ser Arg Leu Asn Ser Ile Arg Asn Ser Phe 545 550 555 560 Leu GlySer Pro Arg Phe His Arg Arg Lys Met Gln Val Pro Thr Ala 565 570 575 GluGlu Met Ser Ser Leu Thr Pro Glu Ser Ser Pro Glu Leu Ala Lys 580 585 590Arg Ser Trp Phe Gly Asn Phe Ile Ser Leu Asp Lys Glu Glu Gln Ile 595 600605 Phe Leu Val Leu Lys Asp Lys Pro Leu Ser Ser Ile Lys Ala Asp Ile 610615 620 Val His Ala Phe Leu Ser Ile Pro Ser Leu Ser His Ser Val Leu Ser625 630 635 640 Gln Thr Ser Phe Arg Ala Glu Tyr Lys Ala Ser Gly Gly ProSer Val 645 650 655 Phe Gln Lys Pro Val Arg Phe Gln Val Asp Ile Ser SerSer Glu Gly 660 665 670 Pro Glu Pro Ser Pro Arg Arg Asp Gly Ser Gly GlyGly Gly Ile Tyr 675 680 685 Ser Val Thr Phe Thr Leu Ile Ser Gly Pro SerArg Arg Phe Lys Arg 690 695 700 Val Val Glu Thr Ile Gln Ala Gln Leu LeuSer Thr His Asp Gln Pro 705 710 715 720 Ser Val Gln Ala Leu Ala Asp GluLys Asn Gly Ala Gln Thr Arg Pro 725 730 735 Ala Gly Ala Pro Pro Arg SerLeu Gln Pro Pro Pro Gly Arg Pro Asp 740 745 750 Pro Glu Leu Ser Ser SerPro Arg Arg Gly Pro Pro Lys Asp Lys Lys 755 760 765 Leu Leu Ala Thr AsnGly Thr Pro Leu Pro 770 775 3 2289 DNA homo sapiens 3 atgggacttgagtttggttt cctagaggct ggtggaaact ggagtcactt cctcccaggg 60 agtgatgggaactggaatct cttcctccca ggaattaatg gaaactggag tctcttcctc 120 ccagggacccatgggaattg gagtctcttt ctcccaagga tcatgggaat tggagttctc 180 tgccaccaggagccagtgga agtgggagac gaggcccttt ggtcctccac atgccccttc 240 cagccctctgccccctctat atcctttagg tacctggttc tggagcacgt ctcggggggt 300 gagctattcgactacctggt aaagaagggg agactgacgc ccaaggaggc ccgaaagttc 360 ttccgccagattgtgtctgc gctggacttc tgccacagct actccatctg ccacagagac 420 ctaaagcccgagaacctgct tttggatgag aaaaacaaca tccgcattgc agacttcggc 480 atggcgtccctgcaggtggg ggacagcctc ctggagacca gctgcgggtc cccccattat 540 gcgtgtccagaggtgattaa gggggaaaaa tatgatggcc gccgggcaga catgtggagc 600 tgtggagtcatcctcttcgc cctgctcgtg ggggctctgc cctttgatga cgacaacctc 660 cgccagctgctggagaaggt gaaacggggc gtcttccaca tgccccactt cattcctcca 720 gattgccagagcctcctgag gggaatgatc gaagtggagc ccgaaaaaag gctcagtctg 780 gagcaaattcagaaacatcc ttggtaccta ggcgggaaac acgagccaga cccgtgcctg 840 gagccagcccctggccgccg ggtagccatg cggagcctgc catccaacgg agagctggac 900 cccgacgtcctagagagcat ggcatcactg ggctgcttca gggaccgcga gaggctgcat 960 cgcgagctgcgcagtgagga ggagaaccaa gaaaagatga tatattatct gcttttggat 1020 cggaaggagcggtatcccag ctgtgaggac caggacctgc ctccccggaa tgatgttgac 1080 cccccccggaagcgtgtgga ttctcccatg ctgagccgtc acgggaagcg gcgaccagag 1140 cggaagtccatggaagtcct gagcatcacc gatgccgggg gtggtggctc ccctgtaccc 1200 acccgacgggccttggagat ggcccagcac agccagagat cccgtagcgt cagtggagcc 1260 tccacgggtctgtcctccag ccctctaagc agcccaagga gtccggtctt ttccttttca 1320 ccggagccgggggctggaga tgaggctcga ggcgggggct ccccgacttc caaaacgcag 1380 acgctgccttctcggggccc caggggtggg ggcgccgggg agcagccccc gccccccagt 1440 gcccgctccacacccctgcc cggcccccca ggctccccgc gctcctctgg cgggaccccc 1500 ttgcactcgcctctgcacac gccccgggcc agtcccaccg ggaccccggg gacaacacca 1560 ccccccagccccggcggtgg cgtcggggga gccgcctgga ggagtcgtct caactccatc 1620 cgcaacagcttcctgggctc ccctcgcttt caccggcgca agatgcaggt ccctaccgct 1680 gaggagatgtccagcttgac gccagagtcc tccccggagc tggcaaaacg ctcctggttc 1740 gggaacttcatctccttgga caaagaagaa caaatattcc tcgtgctaaa ggacaaacct 1800 ctcagcagcatcaaagcaga catcgtccat gcctttctgt cgatccccag cctgagtcac 1860 agtgtgctgtcacagaccag cttcagggcc gagtacaagg ccagtggcgg cccctccgtc 1920 ttccaaaagcccgtccgctt ccaggtggac atcagctcct ctgagggtcc agagccctcc 1980 ccgcgacgggacggcagcgg aggtggtggc atctactccg tcaccttcac tctcatctcg 2040 ggtcccagccgtcggttcaa gcgagtggtg gagaccatcc aggcacagct cctgagcact 2100 catgaccagccctccgtgca ggccctggca gacgagaaga acggggccca gacccggcct 2160 gctggtgccccaccccgaag cctgcagccc ccacccggcc gcccagaccc agagctgagc 2220 agctctccccgccgaggccc ccccaaggac aagaagctcc tggccaccaa cgggacccct 2280 ctgccctga2289 4 762 PRT homo sapiens 4 Met Gly Leu Glu Phe Gly Phe Leu Glu AlaGly Gly Asn Trp Ser His 1 5 10 15 Phe Leu Pro Gly Ser Asp Gly Asn TrpAsn Leu Phe Leu Pro Gly Ile 20 25 30 Asn Gly Asn Trp Ser Leu Phe Leu ProGly Thr His Gly Asn Trp Ser 35 40 45 Leu Phe Leu Pro Arg Ile Met Gly IleGly Val Leu Cys His Gln Glu 50 55 60 Pro Val Glu Val Gly Asp Glu Ala LeuTrp Ser Ser Thr Cys Pro Phe 65 70 75 80 Gln Pro Ser Ala Pro Ser Ile SerPhe Arg Tyr Leu Val Leu Glu His 85 90 95 Val Ser Gly Gly Glu Leu Phe AspTyr Leu Val Lys Lys Gly Arg Leu 100 105 110 Thr Pro Lys Glu Ala Arg LysPhe Phe Arg Gln Ile Val Ser Ala Leu 115 120 125 Asp Phe Cys His Ser TyrSer Ile Cys His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Leu Leu Leu AspGlu Lys Asn Asn Ile Arg Ile Ala Asp Phe Gly 145 150 155 160 Met Ala SerLeu Gln Val Gly Asp Ser Leu Leu Glu Thr Ser Cys Gly 165 170 175 Ser ProHis Tyr Ala Cys Pro Glu Val Ile Lys Gly Glu Lys Tyr Asp 180 185 190 GlyArg Arg Ala Asp Met Trp Ser Cys Gly Val Ile Leu Phe Ala Leu 195 200 205Leu Val Gly Ala Leu Pro Phe Asp Asp Asp Asn Leu Arg Gln Leu Leu 210 215220 Glu Lys Val Lys Arg Gly Val Phe His Met Pro His Phe Ile Pro Pro 225230 235 240 Asp Cys Gln Ser Leu Leu Arg Gly Met Ile Glu Val Glu Pro GluLys 245 250 255 Arg Leu Ser Leu Glu Gln Ile Gln Lys His Pro Trp Tyr LeuGly Gly 260 265 270 Lys His Glu Pro Asp Pro Cys Leu Glu Pro Ala Pro GlyArg Arg Val 275 280 285 Ala Met Arg Ser Leu Pro Ser Asn Gly Glu Leu AspPro Asp Val Leu 290 295 300 Glu Ser Met Ala Ser Leu Gly Cys Phe Arg AspArg Glu Arg Leu His 305 310 315 320 Arg Glu Leu Arg Ser Glu Glu Glu AsnGln Glu Lys Met Ile Tyr Tyr 325 330 335 Leu Leu Leu Asp Arg Lys Glu ArgTyr Pro Ser Cys Glu Asp Gln Asp 340 345 350 Leu Pro Pro Arg Asn Asp ValAsp Pro Pro Arg Lys Arg Val Asp Ser 355 360 365 Pro Met Leu Ser Arg HisGly Lys Arg Arg Pro Glu Arg Lys Ser Met 370 375 380 Glu Val Leu Ser IleThr Asp Ala Gly Gly Gly Gly Ser Pro Val Pro 385 390 395 400 Thr Arg ArgAla Leu Glu Met Ala Gln His Ser Gln Arg Ser Arg Ser 405 410 415 Val SerGly Ala Ser Thr Gly Leu Ser Ser Ser Pro Leu Ser Ser Pro 420 425 430 ArgSer Pro Val Phe Ser Phe Ser Pro Glu Pro Gly Ala Gly Asp Glu 435 440 445Ala Arg Gly Gly Gly Ser Pro Thr Ser Lys Thr Gln Thr Leu Pro Ser 450 455460 Arg Gly Pro Arg Gly Gly Gly Ala Gly Glu Gln Pro Pro Pro Pro Ser 465470 475 480 Ala Arg Ser Thr Pro Leu Pro Gly Pro Pro Gly Ser Pro Arg SerSer 485 490 495 Gly Gly Thr Pro Leu His Ser Pro Leu His Thr Pro Arg AlaSer Pro 500 505 510 Thr Gly Thr Pro Gly Thr Thr Pro Pro Pro Ser Pro GlyGly Gly Val 515 520 525 Gly Gly Ala Ala Trp Arg Ser Arg Leu Asn Ser IleArg Asn Ser Phe 530 535 540 Leu Gly Ser Pro Arg Phe His Arg Arg Lys MetGln Val Pro Thr Ala 545 550 555 560 Glu Glu Met Ser Ser Leu Thr Pro GluSer Ser Pro Glu Leu Ala Lys 565 570 575 Arg Ser Trp Phe Gly Asn Phe IleSer Leu Asp Lys Glu Glu Gln Ile 580 585 590 Phe Leu Val Leu Lys Asp LysPro Leu Ser Ser Ile Lys Ala Asp Ile 595 600 605 Val His Ala Phe Leu SerIle Pro Ser Leu Ser His Ser Val Leu Ser 610 615 620 Gln Thr Ser Phe ArgAla Glu Tyr Lys Ala Ser Gly Gly Pro Ser Val 625 630 635 640 Phe Gln LysPro Val Arg Phe Gln Val Asp Ile Ser Ser Ser Glu Gly 645 650 655 Pro GluPro Ser Pro Arg Arg Asp Gly Ser Gly Gly Gly Gly Ile Tyr 660 665 670 SerVal Thr Phe Thr Leu Ile Ser Gly Pro Ser Arg Arg Phe Lys Arg 675 680 685Val Val Glu Thr Ile Gln Ala Gln Leu Leu Ser Thr His Asp Gln Pro 690 695700 Ser Val Gln Ala Leu Ala Asp Glu Lys Asn Gly Ala Gln Thr Arg Pro 705710 715 720 Ala Gly Ala Pro Pro Arg Ser Leu Gln Pro Pro Pro Gly Arg ProAsp 725 730 735 Pro Glu Leu Ser Ser Ser Pro Arg Arg Gly Pro Pro Lys AspLys Lys 740 745 750 Leu Leu Ala Thr Asn Gly Thr Pro Leu Pro 755 760 52112 DNA homo sapiens 5 atgaaggtgg agcgggagat cgccatcctg aagctcatcgaacacccaca tgtcctcaag 60 ctccacgacg tctacgagaa caagaaatat ttgtacctggttctggagca cgtctcgggg 120 ggtgagctat tcgactacct ggtaaagaag gggagactgacgcccaagga ggcccgaaag 180 ttcttccgcc agattgtgtc tgcgctggac ttctgccacagctactccat ctgccacaga 240 gacctaaagc ccgagaacct gcttttggat gagaaaaacaacatccgcat tgcagacttc 300 ggcatggcgt ccctgcaggt gggggacagc ctcctggagaccagctgcgg gtccccccat 360 tatgcgtgtc cagaggtgat taagggggaa aaatatgatggccgccgggc agacatgtgg 420 agctgtggag tcatcctctt cgccctgctc gtgggggctctgccctttga tgacgacaac 480 ctccgccagc tgctggagaa ggtgaaacgg ggcgtcttccacatgcccca cttcattcct 540 ccagattgcc agagcctcct gaggggaatg atcgaagtggagcccgaaaa aaggctcagt 600 ctggagcaaa ttcagaaaca tccttggtac ctaggcgggaaacacgagcc agacccgtgc 660 ctggagccag cccctggccg ccgggtagcc atgcggagcctgccatccaa cggagagctg 720 gaccccgacg tcctagagag catggcatca ctgggctgcttcagggaccg cgagaggctg 780 catcgcgagc tgcgcagtga ggaggagaac caagaaaagatgatatatta tctgcttttg 840 gatcggaagg agcggtatcc cagctgtgag gaccaggacctgcctccccg gaatgatgtt 900 gacccccccc ggaagcgtgt ggattctccc atgctgagccgtcacgggaa gcggcgacca 960 gagcggaagt ccatggaagt cctgagcatc accgatgccgggggtggtgg ctcccctgta 1020 cccacccgac gggccttgga gatggcccag cacagccagagatcccgtag cgtcagtgga 1080 gcctccacgg gtctgtcctc cagccctcta agcagcccaaggagtccggt cttttccttt 1140 tcaccggagc cgggggctgg agatgaggct cgaggcgggggctccccgac ttccaaaacg 1200 cagacgctgc cttctcgggg ccccaggggt gggggcgccggggagcagcc cccgcccccc 1260 agtgcccgct ccacacccct gcccggcccc ccaggctccccgcgctcctc tggcgggacc 1320 cccttgcact cgcctctgca cacgccccgg gccagtcccaccgggacccc ggggacaaca 1380 ccacccccca gccccggcgg tggcgtcggg ggagccgcctggaggagtcg tctcaactcc 1440 atccgcaaca gcttcctggg ctcccctcgc tttcaccggcgcaagatgca ggtccctacc 1500 gctgaggaga tgtccagctt gacgccagag tcctccccggagctggcaaa acgctcctgg 1560 ttcgggaact tcatctcctt ggacaaagaa gaacaaatattcctcgtgct aaaggacaaa 1620 cctctcagca gcatcaaagc agacatcgtc catgcctttctgtcgatccc cagcctgagt 1680 cacagtgtgc tgtcacagac cagcttcagg gccgagtacaaggccagtgg cggcccctcc 1740 gtcttccaaa agcccgtccg cttccaggtg gacatcagctcctctgaggg tccagagccc 1800 tccccgcgac gggacggcag cggaggtggt ggcatctactccgtcacctt cactctcatc 1860 tcgggtccca gccgtcggtt caagcgagtg gtggagaccatccaggcaca gctcctgagc 1920 actcatgacc agccctccgt gcaggccctg gcagacgagaagaacggggc ccagacccgg 1980 cctgctggtg ccccaccccg aagcctgcag cccccacccggccgcccaga cccagagctg 2040 agcagctctc cccgccgagg cccccccaag gacaagaagctcctggccac caacgggacc 2100 cctctgccct ga 2112 6 703 PRT homo sapiens 6Met Lys Val Glu Arg Glu Ile Ala Ile Leu Lys Leu Ile Glu His Pro 1 5 1015 His Val Leu Lys Leu His Asp Val Tyr Glu Asn Lys Lys Tyr Leu Tyr 20 2530 Leu Val Leu Glu His Val Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val 35 4045 Lys Lys Gly Arg Leu Thr Pro Lys Glu Ala Arg Lys Phe Phe Arg Gln 50 5560 Ile Val Ser Ala Leu Asp Phe Cys His Ser Tyr Ser Ile Cys His Arg 65 7075 80 Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Glu Lys Asn Asn Ile Arg 8590 95 Ile Ala Asp Phe Gly Met Ala Ser Leu Gln Val Gly Asp Ser Leu Leu100 105 110 Glu Thr Ser Cys Gly Ser Pro His Tyr Ala Cys Pro Glu Val IleLys 115 120 125 Gly Glu Lys Tyr Asp Gly Arg Arg Ala Asp Met Trp Ser CysGly Val 130 135 140 Ile Leu Phe Ala Leu Leu Val Gly Ala Leu Pro Phe AspAsp Asp Asn 145 150 155 160 Leu Arg Gln Leu Leu Glu Lys Val Lys Arg GlyVal Phe His Met Pro 165 170 175 His Phe Ile Pro Pro Asp Cys Gln Ser LeuLeu Arg Gly Met Ile Glu 180 185 190 Val Glu Pro Glu Lys Arg Leu Ser LeuGlu Gln Ile Gln Lys His Pro 195 200 205 Trp Tyr Leu Gly Gly Lys His GluPro Asp Pro Cys Leu Glu Pro Ala 210 215 220 Pro Gly Arg Arg Val Ala MetArg Ser Leu Pro Ser Asn Gly Glu Leu 225 230 235 240 Asp Pro Asp Val LeuGlu Ser Met Ala Ser Leu Gly Cys Phe Arg Asp 245 250 255 Arg Glu Arg LeuHis Arg Glu Leu Arg Ser Glu Glu Glu Asn Gln Glu 260 265 270 Lys Met IleTyr Tyr Leu Leu Leu Asp Arg Lys Glu Arg Tyr Pro Ser 275 280 285 Cys GluAsp Gln Asp Leu Pro Pro Arg Asn Asp Val Asp Pro Pro Arg 290 295 300 LysArg Val Asp Ser Pro Met Leu Ser Arg His Gly Lys Arg Arg Pro 305 310 315320 Glu Arg Lys Ser Met Glu Val Leu Ser Ile Thr Asp Ala Gly Gly Gly 325330 335 Gly Ser Pro Val Pro Thr Arg Arg Ala Leu Glu Met Ala Gln His Ser340 345 350 Gln Arg Ser Arg Ser Val Ser Gly Ala Ser Thr Gly Leu Ser SerSer 355 360 365 Pro Leu Ser Ser Pro Arg Ser Pro Val Phe Ser Phe Ser ProGlu Pro 370 375 380 Gly Ala Gly Asp Glu Ala Arg Gly Gly Gly Ser Pro ThrSer Lys Thr 385 390 395 400 Gln Thr Leu Pro Ser Arg Gly Pro Arg Gly GlyGly Ala Gly Glu Gln 405 410 415 Pro Pro Pro Pro Ser Ala Arg Ser Thr ProLeu Pro Gly Pro Pro Gly 420 425 430 Ser Pro Arg Ser Ser Gly Gly Thr ProLeu His Ser Pro Leu His Thr 435 440 445 Pro Arg Ala Ser Pro Thr Gly ThrPro Gly Thr Thr Pro Pro Pro Ser 450 455 460 Pro Gly Gly Gly Val Gly GlyAla Ala Trp Arg Ser Arg Leu Asn Ser 465 470 475 480 Ile Arg Asn Ser PheLeu Gly Ser Pro Arg Phe His Arg Arg Lys Met 485 490 495 Gln Val Pro ThrAla Glu Glu Met Ser Ser Leu Thr Pro Glu Ser Ser 500 505 510 Pro Glu LeuAla Lys Arg Ser Trp Phe Gly Asn Phe Ile Ser Leu Asp 515 520 525 Lys GluGlu Gln Ile Phe Leu Val Leu Lys Asp Lys Pro Leu Ser Ser 530 535 540 IleLys Ala Asp Ile Val His Ala Phe Leu Ser Ile Pro Ser Leu Ser 545 550 555560 His Ser Val Leu Ser Gln Thr Ser Phe Arg Ala Glu Tyr Lys Ala Ser 565570 575 Gly Gly Pro Ser Val Phe Gln Lys Pro Val Arg Phe Gln Val Asp Ile580 585 590 Ser Ser Ser Glu Gly Pro Glu Pro Ser Pro Arg Arg Asp Gly SerGly 595 600 605 Gly Gly Gly Ile Tyr Ser Val Thr Phe Thr Leu Ile Ser GlyPro Ser 610 615 620 Arg Arg Phe Lys Arg Val Val Glu Thr Ile Gln Ala GlnLeu Leu Ser 625 630 635 640 Thr His Asp Gln Pro Ser Val Gln Ala Leu AlaAsp Glu Lys Asn Gly 645 650 655 Ala Gln Thr Arg Pro Ala Gly Ala Pro ProArg Ser Leu Gln Pro Pro 660 665 670 Pro Gly Arg Pro Asp Pro Glu Leu SerSer Ser Pro Arg Arg Gly Pro 675 680 685 Pro Lys Asp Lys Lys Leu Leu AlaThr Asn Gly Thr Pro Leu Pro 690 695 700

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence encoding the amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6.
 2. The isolated nucleic acid moleculeof claim 1, comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:2.
 3. The isolated nucleic acid molecule ofclaim 1, comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:4.
 4. The isolated nucleic acid molecule ofclaim 1, comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:6.
 5. A substantially isolated proteinhaving the kinase activity of the protein shown in SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:6, which is encoded by a nucleotide sequence thathybridizes to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 under highlystringent conditions.